Analysis of single molecules by EM provides a powerful approach to study the mechanics of DNA replication. The continuing focus in this renewal application is how eukaryotic origins are opened by Origin Recognition Complexes (ORC) and how DNA strands are looped at the fork to coordinate leading and lagging strand replication. A statistically large number of molecules are examined and this information combined, with data from biochemical assays. A highly interactive program has been established with Dr. Steve Bell at MIT (yeast ORC), Drs. Tom Broker and Louise Chow at UAB, (human papillomaviruses), Dr. Charles Richard at Harvard (T7 replication), and Dr. Nancy Nossal at the NIH (T 4 replication). Using several EM techniques, the structure of the yeast ORC will be examined alone, and bound to yeast ARS elements. A molecular pointer will localize individual subunits of the ORC, each individually tagged with biotin. EM will be used to explore the unwinding of the ARS by the ORC and other factors. The nature of eukaryotic origin recognition and opening will be further probed using the human papillomavirus. The ability of the viral E1 protein to unwind the origin in a reaction facilitated by E2 and host chaperone proteins will be examined. The role of host DNA polymerase alpha and cellular cyclin E and cdk2 kinase will be explored. The folding of the lagging strand at a moving T7 replication fork will continued to be probed using new mini- circle DNA templates. EM will be used to explore the question of now Okazaki fragment size is controlled and the role of newly discovered novel structural 'spools' created by the binding of single stranded DNA on the lagging strand by the T7 SSB. Using the T4 replication system studies will continue on the looping of the lagging strand in coordinating replication. The nature of the loop and proteins required will be examined. Each system offers a unique window into these questions and information garnered from one is immediately applied to the others. This grant has also provided a unique national resource for the development and application of EM methods for studying DNA and DNA-protein complexes and many NIH-funded investigators in need of help in this technology have been aided.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Wolfe, Paul B
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
Zip Code
Nicholls, Thomas J; Nadalutti, Cristina A; Motori, Elisa et al. (2018) Topoisomerase 3? Is Required for Decatenation and Segregation of Human mtDNA. Mol Cell 69:9-23.e6
Kar, Anirban; Jones, Nathan; Arat, N Özlem et al. (2018) Long repeating (TTAGGG) n single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation. J Biol Chem 293:9473-9485
Amunugama, Ravindra; Willcox, Smaranda; Wu, R Alex et al. (2018) Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading. Cell Rep 23:3419-3428
Zhu, Cheng; Beck, Matthew V; Griffith, Jack D et al. (2018) Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A 115:4661-4665
Erdel, Fabian; Kratz, Katja; Willcox, Smaranda et al. (2017) Telomere Recognition and Assembly Mechanism of Mammalian Shelterin. Cell Rep 18:41-53
Bermek, Oya; Weller, Sandra K; Griffith, Jack D (2017) The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks. J Biol Chem 292:15611-15621
Prasad, Rajendra; Ça?layan, Melike; Dai, Da-Peng et al. (2017) DNA polymerase ?: A missing link of the base excision repair machinery in mammalian mitochondria. DNA Repair (Amst) 60:77-88
Sepsiova, Regina; Necasova, Ivona; Willcox, Smaranda et al. (2016) Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins. PLoS One 11:e0154225
Kar, Anirban; Willcox, Smaranda; Griffith, Jack D (2016) Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops. Nucleic Acids Res 44:9369-9380
Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R et al. (2015) Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. Proc Natl Acad Sci U S A 112:9334-9

Showing the most recent 10 out of 183 publications